R.R. Katkam

Onapristone (ZK 98.299): A potential antiprogestin for endometrial contraception

OBJECTIVES The effects of the antiprogestin onapristone (ZK 98.299) on fertility; menstrual cycle length; duration of menses; serum estradiol, progesterone, and cortisol concentrations; and endometrial morphologic features were studied in adult bonnet monkeys. STUDY DESIGN Five animals were treated subcutaneously with the vehicle and another nine with either 2.5 (n = 4) or 5 mg of onapristone per animal (n = 5). Treatment was initiated on day 5 of the first treatment cycle, and thereafter onapristone was administered every third day for four to seven consecutive cycles. The females were placed with adult males during the periovulatory period, which was assessed by frequent analysis of serum estradiol concentrations. In the final treatment cycle an endometrial biopsy was performed on day 8 after a midcycle estradiol peak in the ovulatory cycle, or around day 20 if the cycle was anovulatory. Blood samples for estradiol, progesterone, and cortisol measurement were collected every third day, except for the periovulatory period when sampling was more frequent. RESULTS Each of the five animals treated with the vehicle became pregnant: one in the first, three in the second, and one in the third mated cycle, whereas only one of nine treated with onapristone became pregnant. Four animals treated with 2.5 mg of onapristone for 17 cycles and another four treated with a 5 mg dose for 21 cycles did not conceive. In eight animals that did not conceive the first three treatment cycles of six were ovulatory, and in the remaining two animals two cycles of each were ovulatory. During treatment the mean menstrual cycle length was not altered significantly; however, in one it was shortened and in another two it was prolonged. Similarly, the mean duration of menses was not significantly affected, but in some cycles it was reduced. Moreover, there was only slight bleeding in some treatment cycles. Ovulation occurred in 30 of 45 treatment cycles, including the final treatment cycle during which the biopsy was taken, as indicated by serum estradiol and progesterone concentrations. In some of the ovulatory cycles prolonged treatment suppressed luteal activity; however, in the ovulatory cycles the duration of follicular and luteal phases was not significantly affected. In the anovulatory cycles there was a delayed increase in serum estradiol concentrations, suggesting a partial inhibition of folliculogenesis. In treated animals endometrial growth and development was retarded and rendered out of phase. In animals treated with the higher (5 mg) onapristone does the endometrial glands had partially regressed, the secretory activity was blocked, and stromal compaction was evident. The treatment had no significant effect on serum cortisol levels. CONCLUSIONS This study demonstrates that low-dose onapristone treatment throughout the menstrual cycle prevents pregnancy without disturbing the menstrual cycle and ovulation in the majority of cycles. However, anovulation and luteal insufficiency occurred in some animals during prolonged treatment. The contraceptive effect in the ovulatory cycles seems primarily related to the retardation of endometrial development resulting in the inhibition of endometrial receptivity. It appears likely that a dose or treatment regimen of onapristone that will inhibit endometrial receptivity and prevent implantation without affecting the menstrual cycle even on prolonged treatment could be identified.

Protein profiling of human endometrial tissues in the midsecretory and proliferative phases of the menstrual cycle

OBJECTIVE To identify the proteins displaying differential expression in midsecretory phase endometrium as compared with proliferative phase endometrium. DESIGN Prospective study with two groups of women in the midsecretory or proliferative phase. SETTING Clinical research outpatient department. PATIENT(S) Healthy, regularly cycling women of proven fertility. INTERVENTION(S) Collection of endometrial biopsy samples. MAIN OUTCOME MEASURE(S) Image analysis software was used to compare two-dimensional protein maps of midsecretory phase endometrial tissues (MSE) with maps of proliferative phase endometrial tissues (PROE) and midsecretory phase uterine fluids (MSU). Matrix-assisted laser desorption/ionization time of flight in tandem (MALDI-TOF-TOF) analysis was carried out to identify eight proteins that were differentially expressed between the two phases and also to identify the spots that shared similar coordinates in the two-dimensional maps of MSE and MSU. RESULT(S) Densitometry analysis and subsequent MALDI-TOF-TOF analysis revealed up-regulation of calreticulin, the beta chain of fibrinogen, adenylate kinase isoenzyme 5, and transferrin in the PROE and of annexin V, alpha1-antitrypsin, creatine kinase, and peroxidoxin 6 in MSE compared with the other phase. Superimposition of the two-dimensional maps of MSE on those of MSU revealed the presence of heat-shock protein 27, transferrin, and alpha1-antitrypsin precursor in both endometrial tissues and uterine secretions. CONCLUSION(S) Differentially expressed proteins identified in the present study could be of relevance in endowing the endometrium with receptivity.

Treatment with a Progesterone Antagonist ZK 98.299 Delays Endometrial Development without Blocking Ovulation in Bonnet Monkeys

The effects of an antiprogestin ZK 98.299 (onapristone) on serum levels of estradiol and progesterone, and on the endometrial morphology were studied in adult bonnet monkeys. Twelve animals having menstrual cycles of normal duration (24 to 30 days) were randomly distributed into 4 equal groups. The animals in Group 1 were treated (s.c.) with the vehicle (benzyl benzoate: castor oil, 1:10), and in Groups 2, 3 and 4 with 5 mg, 10 mg, or 20 mg ZK 98.299 once-a-week, respectively. Treatment was initiated on day 1 of the menstrual cycle and each animal in Groups 1, 2 and 3 was treated for two consecutive cycles. Since the treatment cycle length of animals in Group 4 was considerably prolonged, they were treated for one menstrual cycle only. Endometrial biopsy was taken around day 20 of the second treatment cycle of first three groups and around day 50 of the 4th group of animals. Treatment with vehicle or 5 mg ZK 98.299 had no significant effect on the menstrual cycle length. Treatment with 10 mg dose had no effect in two animals and prolonged the cycle length in one, whereas, further increase in the dose to 20 mg prolonged the cycle length in all the animals. The duration of menses was generally reduced. Treatment with vehicle or different doses of ZK 98.299 had no effect on ovulation. In animals treated with 5 or 10 mg dose, the pattern of mid cycle rise in serum estradiol levels and progesterone levels during the luteal phase of both treatment cycles were comparable to those of vehicle-treated animals and were suggestive of normal ovulatory cycles. On the other hand, in animals treated with the higher dose (20 mg/week), progesterone levels during the luteal phase were significantly reduced and were indicative of luteal insufficiency. The hormonal data during the treatment period of this group of animals was suggestive of two distinct ovarian cycles indicating that the menstrual bleeding during the treatment period was probably very scanty. Treatment with ZK 98.299 impaired the endometrial development in a dose-dependent manner. In vehicle-treated animals, the endometrium had large and tortous glands with secretions. Treatment with ZK 98.299 caused atrophic changes in the glands as well as in the stroma. The height of the epithelial cells was markedly decreased and they became small and inactive. This study, therefore, suggests that treatment with low doses of antiprogestin ZK 98.299 at weekly intervals does not block folliculogenesis or ovulation, but has an inhibitory effect on the endometrium.(ABSTRACT TRUNCATED AT 250 WORDS)

Endometrial contraception: modulation of molecular determinants of uterine receptivity

Modulation of endometrial receptivity is a promising approach for fertility regulation since it allows a contraceptive to act specifically at the endometrium. This was corroborated by our previous observations that treatment with low doses of a pure progesterone antagonist (PA, antiprogestin), onapristone (ZK 98299), in bonnet monkeys inhibited fertility by selectively retarding endometrial development, without affecting the hypophyseal-hypothalamic function. In the present study, further investigations, undertaken to analyze the molecular repertoire of a nonreceptive primate endometrium, determined expression of: steroid hormone receptors, i.e. progesterone receptor (PR) and estrogen receptor (ER); cytokines, i.e. leukemia inhibitory factor (LIF): transforming growth factor beta (TGFbeta) and its receptor (TGFbetaR); and cell adhesion molecules, i.e. integrins (alpha(v)beta(3), alpha(1)beta(1)). These studies were conducted during the different phases of the normal menstrual cycle and following treatment with different doses of onapristone (2.5 mg, 5 mg, or 10 mg every third day for one cycle) in bonnet monkeys. The molecules were analysed collectively to explore the possibility of a correlation between expression of these markers and endometrial receptivity and to investigate whether there exists a regulatory link between expression of these molecules under in vivo conditions. Three types of expression patterns of endometrial factors were observed during the peri-implantation period following onapristone treatment: 1) LIF, alpha(v)beta(3), and alpha(1)beta(1) showed significant (P < 0.02) down regulation in glandular epithelium of endometria in animals treated with all three doses of onapristone as compared to the control group. This was indicative of their critical role in the progesterone-driven cascade leading to implantation. 2) PR, TGFbeta, and TGFbetaR remained unaffected in the endometria from 2.5 mg treated animals and showed down regulation in animals treated with 5 and 10 mg onapristone as compared to the control group, thereby suggesting that the expression of these markers may not truely reflect endometrial receptivity per se. However, their facilitatory role in preparing the endometrium for implantation can not be ruled out since continued perturbation in the expression of these molecules may affect endometrial growth, remodelling, and differentiation, which in turn may render the endometrium nonreceptive; 3) ER remained unaltered in endometria of animals rendered infertile with 2.5, 5, and 10 mg onapristone. This observation indirectly suggests that onapristone-induced endometrial changes are mediated via some specific mechanisms. The present study clearly demonstrates that endometrial non-receptivity induced at low doses of onapristone is associated with changes in the expression pattern of specific molecular markers. However, no direct correlation was observed between in vivo expression of TGFbeta, LIF, and integrins, thereby lending support to the concept that there exists redundancy or multiple pathways which regulate implantation events.

Effects of an Antiprogestin Onapristone on the Endometrium of Bonnet Monkeys: Morphometric and Ultrastructural Studies

Abstract Our previous studies demonstrated the ability of low doses of antiprogestin ZK 98.299 (onapristone) to inhibit fertility in bonnet monkeys. In the present study cumulative effects of low doses of ZK 98.299 on the endometrial cytoarchitecture of bonnet monkeys were analyzed. Treatment with either the vehicle (n = 3) or onapristone at 2.5 mg (n = 4) or 5.0 mg (n = 3) was initiated on Day 5 of the first menstrual cycle and thereafter repeated every third day for four to seven consecutive cycles. The last treatment cycles were anovulatory in two animals treated with 2.5 mg and all animals treated with 5.0 mg. Endometrial biopsies were collected on Day 8 after the midcycle estradiol peak in ovulatory menstrual cycles and on Day 20 in anovulatory menstrual cycles during the last treatment cycle. Ultrathin sections of the fixed endometrium were stained with toluidine blue for morphometric analysis and uranyl acetate and lead citrate for ultrastructural analysis. The ZK 98.299-treated animals showed a dose-dependent endometrial atrophy as evident by a decrease in the height and diameter of the glands and early signs of compaction in the stroma. Ultrastructural analysis also revealed dose-dependent degenerative changes in the subcellular organelles such as the nucleus, mitochondria, endoplasmic reticulum, lysosomes, and Golgi apparatus. This suggests that long-term treatment with low doses of ZK 98.299 leads to the suppression of estrogen-dependent endometrial proliferation. However, this blockade operates independent of estradiol receptor (ER) and progesterone receptor (PR) concentrations as the expressions of these steroid receptors did not show any significant changes even after prolonged treatment. The study demonstrated an antiestrogenic effect of ZK 98.299 on endometrium after prolonged treatment in bonnet monkeys.

High mobility group box 1 (HMGB1) protein in human uterine fluid and its relevance in implantation

STUDY QUESTION Does a differential abundance of high mobility group box 1 (HMGB1) protein in uterine fluid (UF) have a functional significance? SUMMARY ANSWER In rats, an excess of HMGB1 in UF during the receptive phase is detrimental to pregnancy. WHAT IS KNOWN ALREADY The identification of constituents of the human uterine secretome has been a subject of renewed interest, due to the advent of high throughput proteomic technologies. Proteomic-based investigations of human UF have revealed the presence of several proteins such as mucins, host defense proteins S100, heat shock protein 27 and haptoglobin, etc. The present study reports on the presence of HMGB1, a nuclear protein, in human UF. Activated macrophages/monocytes, natural killer cells, mature dendritic cells, pituicytes and erythroleukemic cells are also known to secrete HMGB1. Existing data suggest that extracellular HMGB1 plays a role in inflammation. STUDY DESIGN, SIZE, DURATION The human part of this study was cross-sectional in design. UF and endometrial tissues were collected from regularly cycling women in the early secretory (i.e. pre-receptive phase, Day 2 post-ovulation, n = 7) or secretory phase (i.e. receptive phase, Day 6 post-ovulation, n = 7) of their menstrual cycles. Samples were also collected from cycling rats in the proestrous (n = 8) or metestrous (n = 8) phase of their estrous cycles. Uteri were also collected from HMGB1-treated pregnant (n = 7) and untreated pseudo-pregnant (n = 7) rats and from pregnant rats at Day 3-5 post-coitum (p.c.) (n = 18, 3 each for six-time points). PARTICIPANTS/MATERIALS, SETTING, METHODS In each group of human samples, four samples were used for isobaric tag for relative and absolute quantification (iTRAQ) analysis and three samples were used for immunoblotting experiments to determine the abundance of HMGB1 in pre-receptive and receptive phase UF samples. HMGB1 levels in rat UF and endometrial tissue samples were estimated by ELISA and immunohistochemical studies, respectively. The expression of inflammation-associated molecules, such as nuclear factor kappa B (NFκB), receptor for advanced glycation end products (RAGEs), tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), was analyzed by immunohistochemistry in HMGB1-treated and pseudo-pregnant rats. MAIN RESULTS AND THE ROLE OF CHANCE HMGB1 was identified as one of the differentially abundant proteins in the list generated by 8-plex iTRAQ analysis of receptive and pre-receptive phase UF samples. In both humans and rats, secreted and cellular levels of HMGB1 showed a similar pattern, i.e. significantly (P < 0.05) lower abundance in the receptive phase compared with that in the pre-receptive phase. A significant (P < 0.05) decline was also observed in the endometrial expression of HMGB1 on the day of implantation in pregnant rats. Exogenous administration of recombinant HMGB1, on Day 3 p.c., led to pregnancy failure, whereas administration of recombinant leukemia inhibitory factor or saline had no effect on pregnant rats. Further investigations revealed morphological changes in the endometrium, an increase in the expression of luminal epithelial NFκB and significantly (P < 0.05) higher expression levels of endometrial RAGE, TNF-α and IL-6 in HMGB1-treated rats, compared with untreated pseudo-pregnant rats. LIMITATIONS, REASONS FOR CAUTION The mechanisms, contributing to a decline in the cellular and extracellular levels of HMGB1 during the receptive phase, remain to be ascertained. WIDER IMPLICATIONS OF THE FINDINGS An excess of HMGB1 in the UF may be associated with infertility in women.

Secrets of Endometrial Receptivity: Some Are Hidden in Uterine Secretome

Endometrium, the innermost mucosal layer of the uterus, serves as a lodge for the embryo in eutherian mammals. The endometrium is constituted of various cell types, and each cell type executes specific functions to facilitate embryo implantation and development. It is well established that the endometrium, despite being non‐permissive to the embryo for the major period of a menstrual cycle, is irreplaceable in the scheme of events essential for procreation. However, the embryo, before initiating physical contact with the endometrium, encounters the uterine cavity that remains bathed in uterine fluid. Uterine fluid is an admixture of endometrial secretions, plasma transudates, and oviductal fluid. Uterine fluid components are believed to play important roles in immunosuppression and embryo development during peri‐implantation period. Uterine fluid is also involved in defense against pathogens, sperm migration, and lubrication of endometrium. The advent of high‐throughput functional genomics tools has created enormous opportunities to investigate the uterine fluid for its protein repertoire and modulation during the receptive phase of an endometrial cycle in animals and humans. Towards this, few investigations have been conducted in recent years. The data obtained using non‐targetted functional genomics approaches need to be assimilated with the existing information on specific components of uterine fluid.

Embryo-induced alterations in the molecular phenotype of primate endometrium.

Reproductive biomedicine has made significant advances in the area of assisted reproductive technologies in the last two and half decades. However, embryo implantation remains a major obstacle in securing high pregnancy rates. Various non-human primate models including rhesus, marmoset and baboon have been employed to elucidate in vivo mechanisms underlying the uterine events that initiate, sustain and complete implantation. This review collates the information available on the molecular profile of gestational endometrium in primates. Collectively, these studies reveal dynamic spatio-temporal changes in the expression of cytokines, growth factors, cell-adhesion molecules, cytoskeleton elements and other factors in the endometrium during the post-implantation phase of pregnancy. Considering that the endometrial events during the pre-implantation stages of pregnancy may dictate implantation success, we have developed a bonnet monkey (Macaca radiata) model where pregnancy can be detected at the pre-implantation stage. Using this model, we investigated some of the endometrial events that occur before the completion of implantation. Remarkable changes were observed in endometrial expression of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNFalpha), as well as expression of immunosuppressive factors such as transforming growth factor beta-2 (TGFbeta2), interleukin-6 (IL-6) and placental protein-14 (PP-14), even before the embryo starts invading the endometrium. This highlights the super-imposition of endometrial receptivity by embryonic stimuli, marked by differential expression and/or localization of the factors that regulate endometrial transformation for embryo survival, growth and development.

Uterine secretome and its modulation in rat (Rattus norvegicus)

The present study identifies uterine fluid (UF) proteins that display differential abundance during the embryo-permissive phase in nonconception and conception cycles in rats. UF samples were collected from nonpregnant rats in the proestrous (n=17) and metestrous (n=18) phases and also from pregnant (n=17) and pseudopregnant (n=17) rats on day 4 post coitus. UF protein profile in the metestrous phase was compared with that in the proestrous phase. Similarly, UF protein profile of the pregnant rats was compared with that of the pseudopregnant rats. Two-dimensional PAGE, followed by densitometric analysis of the paired protein spots, revealed differential abundance of 44 proteins in the metestrous phase, compared with that in the proestrous phase. Of these, 29 proteins were identified by matrix-assisted laser desorption/ionization time-of-flight or liquid chromatography-tandem mass spectrometry. Functional groups such as proteases, protease inhibitors, and oxidoreductases were enriched in differentially abundant proteins. Total protease activity in UF was found to be significantly (P<0.05; t-test) higher in the proestrous phase, compared with that in the metestrous phase. Furthermore, 41 UF proteins were found to be differentially abundant in pregnant rats, compared with pseudopregnant rats. Of these, 11 proteins could be identified. Immunoblotting analysis confirmed significantly higher (P<0.05; t-test) abundance of β-actin, Rho-specific guanine nucleotide dissociation inhibitor alpha (Rho-GDIα), and peroxiredoxin-2 and -6 in the metestrous phase, compared with that in the proestrous phase. Compared with pseudopregnant rats, pregnant rats had significantly higher (P<0.05; t-test) levels of UF β-actin and Rho-GDIα. Furthermore, these proteins could be detected in the culture supernatants of endometrial epithelial cell lines, thereby providing an evidence of their secretion from endometrial epithelial cells. Data obtained from the study expand our knowledge on the uterine milieu that favours embryo implantation.

Differential expression of calreticulin, a reticuloplasmin in primate endometrium

BACKGROUND To our knowledge, there are no data on hormonal regulation of reticuloplasmins in primate endometrium. We report the presence and modulation of expression of three reticuloplasmins in endometrium of bonnet monkeys (Macaca radiata). METHODS Receptive and non-receptive endometria obtained from vehicle-treated control and onapristone (antiprogestin)-treated animals, respectively, were compared for differentially expressed proteins by two-dimensional proteomics. Mass spectrometric analysis annotated two such proteins as calreticulin and protein disulfide-isomerase (PDI), known to be molecular chaperones in endoplasmic reticulum. We then investigated if endoplasmin, another reticuloplasmin is also differentially expressed. Expression of these reticuloplasmins was also investigated in the endometriuma during pregnancy in bonnet monkeys. Samples were analysed by immunohistochemistry and western blot (calreticulin in human endometrium), and calreticulin transcript levels in Ishikawa cell line were assessed by real time PCR. RESULTS Immunohistochemical analysis of the functionalis region of non-receptive endometria in monkeys revealed higher expression of (i) calreticulin (P < 0.01) in glandular epithelium and (ii) PDI in stroma (P < 0.0001), but no change in endoplasmin in stroma or glands, compared with receptive endometria. Protein level of all three reticuloplasmins in the stromal region of endometrial functionalis was higher in pregnant than non-pregnant animals (P < 0.05). Human endometrial calreticulin protein was higher in the estrogen-dominant (proliferative) phase than progesterone-dominant (mid-secretory) phase of the cycle. Calreticulin mRNA in Ishikawa cells is up-regulated by estrogen (P < 0.05 versus control), with a trend towards down-regulation by progesterone. CONCLUSION Our data suggest that endometrial reticuloplasmins are regulated by hormones and embryonic stimuli in a cell-type specific manner. These novel data open up new lines of investigation for elucidating the mechanisms by which hormones or embryonic stimuli influence the sub-cellular physiology of endometrium.